Measuring Diffusion in a Living Cell Using Fluorescence Correlation Spectroscopy. A Closer Look at Anomalous Diffusion Using HIV-1 Integrase and its Interactions as a Probe

Abstract

Fluorescence correlation spectroscopy is a powerful technique for observing the diffusion offluorescent molecules. In buffer solution the autocorrelation analysis of the intensity fluctuations allowsthe calculation of the diffusion coefficient, and therefore the size of the molecule, as well as its interactions.In living cells, however, the situation is more complicated because the viscosity of the medium varies a lotfrom spot to spot. Moreover, the analysis of the autocorrelation curve is not always straightforward andthe chosen analysis method can sometimes be questioned. Autocorrelation curves obtained from living cellscan be fitted equally well with a multicomponent model or with an anomalous diffusion model (Wachsmuthet al., J Mol Biol, 298:677, 2000). The latter model introduces a global anomaly parameterthat describes the heterogeneity of the hindrance of the medium to the diffusing molecules in the cell.By applying the two models on a system of interacting proteins in living cells, we could compare theadvantages and disadvantages of both models. This article will give an introduction to fluorescence correlationspectroscopy as well as HIV-1 integrase, whose protein fragments have been used as a model in thisstudy. HIV-1 integrase has been shown to interact with a cellular protein, called lens epithelium-derivedgrowth factor or LEDGF/p75. This interaction will be used as a starting point for the comparison ofthe two models.